High industrial value is widely recognized in the production of substances from animal cells including human cells. They include such substances with wide use for medicine and reagenets as viral antigenic proteins from virus-infected cell, interferons, cytokines, growth factors, monoclonal antibodies, and tissue plasminogen activator.
In addition, mass cells themselves, such as cultured skin keratinocytes, endothelial cells, liver cells and pancreatic Langerhans cells are of great value, since they have successfully been applied in therapy in such ways or forms as skin grafting, hybrid-type artificial blood vessels, artificial organs and transplantation in experimental level. A particular example of success in experimental level is the application of a mass culture of liver cells in the replacement of liver function: A mass culture of liver cells in a vessel, which is connected to a living body from outside through artificial blood vessel, works as an extracorporeal liver, and is expected to overcome liver disfunction in an acute stage.
Technological needs therefore exist in the methods and devices to obtain a mass culture of such bio-organisms as animal cells including human cells, as well as to maintain cultured cells for a long time in a high cell density.
Below, related art is described exemplifying the methods of animal cell culture with the purpose of substance production.
(1) Stationary monolayer culture method.
In this method, cells are cultivated in the state that cells adhere, either actively or passively, to the surface of flat substratum, such as glass and plastics. Plastic culture bottles have widely been used. The use of culture vessels, each with a large surface of multi-layered plastic plates settled in a cuboid module, is the representative technique of mass cell culture (Weiss, R. S. & Schleicher, J. B., Biotech. Bioeng. 10:601-616 (1968). The technique using this type of culture vessel is generally operated with batch culture method, i.e., a method in which cells inoculated in a vessel are fed with culture medium for a period of time, and then the culture is terminated yielding cells and spent medium. Although being the most basic method of mass cell culture for adherent cells, this method has such restriction that the number of cells cultivated is relatively small per volume of culture vessel, that man power for handling culture vessels and feeding cells is large, and that a culture vessel is costive, if it is disposable.
(2) Movable monolayer culture method
In this method, a culture vessel is moved, or rotated while cells grow in a monolayer. This method partially solved problems associated with the stationary monolayer culture method. Using various mechanical means, feeding cells with gas and nutrient has become efficient. The area of substrate surface can be increased extensively. This method is exemplified by a technique (Roller bottle culture method) in which the cell monolayer is formed in the internal surface of a rolling cylindric culture bottle (roller bottle). A number of modifications of the roller bottle culture method have been attempted in order to increase cell density per device volume. In one example, a sheet of wavy plastic thin plate and a sheet of flat plastic thin plate are alternatively rolled in within the interior of the roller bottle (Sterilin, Bulk Culture Vessel, Sterilin Ltd., Teddington, Middlesex).
The movable monolayer culture method is for increasing the contact of cells with culture medium and gas phase by rotating the culture device itself, or culture substratum, and thus has no relation to the present invention, which essentially is a culture method for a flowing culture solution with the substratum fixed.
(3) Micro-carrier culture method.
In this method, cells are made attached onto, or penetrated into micro-particles, which are suspended in liquid medium by mechanical force. Thus, cells are cultivated while they are attached on or in the floating micro-particles in suspension. Many kinds of micro-particle carriers (micro-carrier) have been devised for use in this technique. Particular attention has been given to developing micro-carriers, suitable for weakly adhesive cells to attach to and for cells to penetrate (Van Wezel, A. L., Nature, 216:64-65 (1967), Van Wezel, A. L. in Animal Cell Biotechnology, ed. by Spier, R. E. & Griffith, J. B., Academic Press, London, Vol. 1, 265-282 (1985)).
A vessel of micro-carrier suspension provides a surface area for cell adhesion much larger than a vessel of either the stationary or movable monolayer culture. With this advantage, the micro-carrier culture method becomes a standard technique for mass cultivation of adherent cells. To maintain micro-carrier particles in a suspended state, impellers, rotators, and the introduction of floating bubbles are used. While representative of methods for mass cultivation which are universally used for adherent cells, despite its disadvantages in the difficulty in keeping micro-carriers in a homogeneous suspension or in damages against cells, the micro-carrier culture method the Press has no relation to invention: In the micro-carrier culture method, the substratum itself is floated and agitated: However, in the present invention, although the micro-carrier can be used as substratum, only the liquid medium is flowed.
(4) Cell culture method using hollow fiber.
In this method a device similar to artificial kidney for dialysis use is employed. The device is composed of a bundle of many tubular semipermeable threads or hollow fibers in a columner case.
Cells are locked in the space between the hollow fibers, while liquid medium is circulated in the semipermeable hollow fiber in order to supply nutrient to the cells and to make gas exchange through the tubular thread. Thereby, a cell density near to that in vivo can be realized (Knazek, R. A., Gullino, P. M. Kohler, P. O. & Science, 178:65-66 (1972), Tyo, M. A., Bulbulian B. J., Menken B. Z., Murphy, T., J. Animal Cell Biotech. 3:357-371 Academic Press, London (1988)).
In addition, cells non-adhering to the hollow fiber can be cultivated, since they can simply be stacked between bundles of filers. Although being modern and frequently used, the hollow fiber culture method has no relation to the present invention. In the hollow fiber culture method, the essential technique is characterized by the compartment for cells and the compartment for circulating liquid medium being separated by the semi-permeable membrane of capillary tubular thread. Although hollow fibers can be used as substratum for the present invention, other substratum can also be used regardless of the semi-permeability of the fibers.
(5) Immobilized cell culture method.
In this method, the feeding of cells with a nutrient medium, gas exchange, and the removal of waste products are performed by a flowing liquid. The cells are immobilized in or on the surface of a substratum, which permits cells to adhere, or the medium to permeate. Since such common products as matrix glass fiber and ceramic monolith can be used as substrata, and since weakly adhesive cells can be immobilized, this method provides a wide range of application.
Among techniques belonging to this culture method, widely used is that for a perfusion culture equipment (Opticell.RTM. culture equipment). In the Opticell.RTM. culture system, medium flows through a pump into a jacket with a porous ceramic module incorporated (Opticore.RTM., in which cells are immobilized. The medium flowing out from the module is further circulated through the channels of a gas exchanger, medium reservoir, monitoring devices, etc. (Lydersen, B. K., Pugh, G. G., Paris, M. S., Sharma, B. P., & Noll, L.A.P. Biotechnology 3: 63-67 (1985)).
The present invention falls under the category of immobilized cell culture method in general. However, it has no relation to the prior arts including the Opticell.RTM. culture method. That is, the present invention is concerned with a type of culture device and related techniques of a culture method. While it is capable of incorporating all materials including porous ceramic module, glass fiber etc. after being worked into the water-permeable and cell-immobilizable form as substratum, it does not target to specify substratum.
(6) Suspension culture method.
In this method, weakly adherent cells are cultivated in a floating state in a liquid medium. In principle, the method is identical to suspension culture of mirco-organisms. For the case using animal cells, various devices have been installed to prevent damages to cells caused by shearing force which happens during agitating culture medium. The method is widely used for modern mass culture. The method, however, has no relation to the present invention which is generally in the category of immobilized cell culture.
The conventional cell culture methods described above are associated with problems described below.
(1) Cell damage.
To maintain cells in a floating state, mechanical force is applied from outside, which inevitably creates a shearing force on the cells. This shearing force gives rise to critical problems in suspension cultures, or micro-carrier cultures of animal cells which lack a cell wall, and are easily damaged by shearing force. This damage becomes a critical factor preventing scaling up of culture volume, and in enhancing cell density.
(2) Long-term maintenance of a culture.
Long-term maintenance of a culture is frequently difficult due to the wear and tear of the culture vessel, substratum and mechanical elements, and due to the changes of culture environment along with the accumulation of cellular substances and/or dead cells.
Actual problems along with a long term culture are as follows: In monolayer cultures, cell layers peel-off. In suspension cultures, cell separation apparatus and gas exchange apparatus clog with aggregates of cells. In micro-carrier cultures, micro-carriers flocculate, and cells exfoliate. In perfusion cultures such as hollow fiber cultures and immobilized cell cultures, increasing load to pumps often causes an unexpected shut down.
(3) Generality of culture methods.
Conventional culture methods are frequently applied only to restricted cell species depending on the existence, non-existence, or the strength of cell adherence. For example, the micro-carrier culture method and movable monolayer culture method require strong adherence of cells to the substratum. On the contrary, adherent cells can not usually be cultivated in suspension culture.
(4) Gas exchange
The efficiency of gas exchange in supplying oxygen for consumption and in removing carbon dioxide produced during cellular metabolism is a critical factor limiting the density of cultured cells.
In fact, in the stationary monolayer culture method, low efficiency in aeration on the surface of the substratum is one of the major critical factors limiting cell multiplication.
In suspension cultures, introduction of the technique for sparging air bubbles in a liquid medium has increased the efficiency of gas exchange greatly. This technique, however, also introduced some problems when applied to animal cell cultures: At the surface of air bubbles, medium proteins tend to denature and cells are damaged.
These problems have been partially solved by introducing a method to supply gas through gas-permeable tubes arranged in the culture medium.
It is now possible with gas-permeable tubes to exchange gas with a very high efficiency. In a long term cell culture, however, gas-permeable tubes should frequently be changed since gas exchange efficiency decreases due to the clogging of the surface of the tubes.
(5) The separation of cells from liquid culture medium and the supply of medium nutrients.
Cell separation in suspension culture method and micro-carrier culture method is carried out with natural precipitation, centrifugation or membrane separation. In a large scale cell culture, cell separation devices are introduced to remove waste medium, and to collect cells or cell-adhered micro-carriers, utilizing differences in density between cells, or cell-adhered micro-carriers and the culture medium.
The separation of cells from liquid culture medium is usually not a problem for stationary monolayer culture method or a culture method with the state of cells adhered to substratum. In contrast, with the suspension cell culture method or the micro-carrier culture method, the clogging and decreasing performance of separation apparatus are important matters. With modern systems advancing into those permitting high cell density, where high performance and long life separation apparatus are required, cell separation methods are thus becoming very important.
(6) Effect on purification process.
In a cell culture system in which a cell product is discharged into culture medium, the load on the purification step varies considerably according to the kind of substances other than the product contained in culture medium. In a culture system which causes a considerable damage to cells, for example, large amounts of proteins and DNA are discharged from damaged cells into the culture medium, causing an increase in the load on the purification step.
(7) Reliability of culture equipment.
With the advancement in performance of cell culture equipment, more and more parts are becoming needed for the construction and operation thereof. In addition to such main parts as a culture vessel and substratum for the cell culture, there are a number of parts, some of which are mentioned below, for example: rotators, stirrers, impellers or vibrators, pumps and their driving parts, cocks, tubes, removable connectors for mechanical parts; and oxygen electrodes, pH electrodes, carbonic acid gas electrodes, pressure sensors, and temperature sensors for monitoring parts.
In addition to these parts, there are gas supply apparatus, condensers and filters for exhausted gas, suppliers of constant temperature water, suppliers of desterilized liquid medium, sterilizing apparata for systems, and automated control installation to combine total systems.
Complication of the system is inevitable. Accordingly, associated problems, i.e., the high cost of culture equipment, the decrease in reliability in mechanical terms, and the insecurity of long term operation, are posed as decisive factors with the modern large scale cell culture systems.
(8) Economy of cell culture
With the purpose of substance production by cell culture, important factors in terms of economy are in the reliability, the cost and the scale of culture equipment. Call density achieved is also an important matter. For reference, the maximum density of cells achieved is as follows: In conventional suspension cultures or stationary monolayer cultures, it is in the order of n.times.10.sup.6 cells/cm.sup.3 ; in micro-carrier cultures, it is in the order of n.times.10.sup.7 cells/cm.sup.3 ; in hollow fiber cultures, it is close to 10.sup.8 /cm.sup.3. An achievement of high density near 10.sup.8 cells/cm.sup.3 is a current target, since there is a competitive alternative choice of production system, i.e. in vivo transplantation of hybridoma cells into animals.